While optical aberrations can be measured and compensated by using adaptive optics (AO) methods 10, 11, previous implementations were predominantly based on 2PM and thus were limited in their effective imaging depth to at most 800 µm in the mouse brain 12, 13, 14, 15. This has so far prohibited the resolution of fine neuronal processes, synapses and subcellular Ca 2+ transients in deep cortical and subcortical areas of the mouse brain in vivo without using highly invasive methods such as gradient index lens implantation 8 or cortical aspiration 9. Imaging approaches based on three-photon excitation microscopy (3PM) have shown potential for deep imaging beyond 1 mm with cellular resolution, owing to a substantially increased signal-to-background ratio (SBR) at depth and longer wavelength excitation 4, 5, 6, 7.Īs in 2PM, however, with increasing imaging depths optical aberrations due to tissue heterogeneities and refractive index mismatches, and subtle motion artifacts due to the animal’s heartbeat, degrade image resolution and overall performance, and result in a loss of subcellular details. However, the maximum penetration depth of two-photon microscopes is fundamentally limited by the onset of out-of-focus fluorescence near the surface with increasing excitation power, which for the mammalian brain prevents imaging beyond roughly 1 mm (ref. In scattering tissues such as the mammalian brain, two-photon excitation microscopy (2PM) is the gold standard for recording cellular structure and function in noninvasive and physiologically relevant conditions in vivo 1, 2.
In addition, we show applications to deep-layer calcium imaging of astrocytes, including fibrous astrocytes that reside in the highly scattering corpus callosum. We demonstrate near-diffraction-limited imaging of deep cortical spines and (sub)cortical dendrites up to a depth of 1.4 mm (the edge of the mouse CA1 hippocampus).
Our modal-based, sensorless AO approach is robust to low signal-to-noise ratios as commonly encountered in deep scattering tissues such as the mouse brain, and permits AO correction over large axial fields of view. Here we describe a minimally invasive intravital imaging methodology based on three-photon excitation, indirect adaptive optics (AO) and active electrocardiogram gating to advance deep-tissue imaging.
However, tissue scattering, optical aberrations and motion artifacts degrade the imaging performance at depth. Multiphoton microscopy has become a powerful tool with which to visualize the morphology and function of neural cells and circuits in the intact mammalian brain.
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